Pcr-based Evaluation Of Prednisolone-induced Relapse Of Asymptomatic Toxoplasma Gondii Infection And The Therapeutic Efficacy Of Azithromycin

Toxoplasma gondii is a kind of worldwide distribution and obligate intracellular protozoan parasite. It is also an important zoontic and veterinary pathogen. Toxoplasma gondii infection is very common in many health people without asymptomatic or only fever, diarrhea and other non-specific symptoms, but Toxoplasma gondii can cause miscarriage, premature birth, stillbirth, deformities and other adverse pregnancy outcomes in pregnancy infection stage. Seriously, Toxoplasma gondii even causes fatal harm in immune suppression or immune deficient people.The outcome of Toxoplasma gondii infection is decided by the interaction between the host and parasite. The harm to host is correlated with the immune state of the host and the cell-mediated immunity which is regarded as the predominant mechanism to resist Toxoplasma gondii infection is the major protective immunity to resist the Toxoplasma gondii infection. It plays an important role in inhibiting the proliferation of parasite, preventing the activation of the cyst, cleaning up the parasite and controlling parasitemia.It is difficult to that is removed completely completely Toxoplasma gondii in health peaple, and it soon to form cysts, which hibernated in tissue. This is known as a kind of latent Toxoplasma gondii infection without asymptomatic. Once the immune function is abnormal, latent Toxoplasma gondii cysts rupture leads to toxoplasmosis recrudescence. In recent years, with the increase of cancer patients and AIDS patients and organ transplant recipients, the number of toxoplasmosis patients gradually on the increase. Increase in these patients and the increase in incidence of toxoplasmosis, maybe there is connection between the two and how the mechanism of these intrinsic link? Interestingly, we found that these patients all are used the hormone during routine treatment. As we all know, on the one hand, hormone can treat the diseases; on the other hand it can also suppress immune system function. Therefore, latent infection of Toxoplasma gondii in mice induced by hormone relapse and therapeutic efficacy of Azithromycin on the basis of assessment for the theory has become extremely important.At first, referring to and improving the previous'method, we purified Toxoplasma gondii cyst from mice'brain tissue and improved its methods. After that, comparison of Lysis buffer extraction method and DNA extraction kit method pros and cons, we selected the method of extraction DNA. Then, used of Toxoplasma gondii B1 gene and AF146527 gene sequence designed two pairs of primers and compared their'sensitivity, we determined to use which one. Soon, a model of infection of Prugniaud strain Toxoplasma gondii in mice established, we defined that the mice how weeks after infection entered into latent infection state. Finally, through injection with prednisolone into hind medial subcutaneous and with Azithromycin intraperitoneally, we evaluated Prednisolone-induced Relapse of Asymptomatic Toxoplasma gondii infection and the therapeutic efficacy of Azithromycin by PCR results and mouse mortality results and the number of brain cysts results.Objective:To establish a model of infection of Prugniaud strain Toxoplasma gondii, then to study PCR-based Evaluation of Prednisolone-induced Relapse of Asymptomatic Toxoplasma gondii Infection and the Therapeutic Efficacy of Azithromycin.Methods:1, Referring to and improving the previous methods, we purified Toxoplasma gondii cyst with the separating medium of lymphocyte by high speed centrifugation, then we evaluate the purified cyst inoculating susceptible mice.2, ICR strain mice were infected intra-abdominally with 10 cysts of Toxoplasma gondii ,then we observed the condition, life spans and pathology feature of infected mice.3, Respectively, used of Toxoplasma gondii B1 gene and AF146527 gene sequence designed two pairs of primers and compared their sensitivity, we determined to use which one4, Respectively, we used Lysis buffer method and DNA extraction kit method to extract DNA from whole blood of mice Toxoplasma gondii infection in the acute stage.5, Female ICR mice, about 20g, were challenged at week 0 with 10 cysts of prugniaud strain Toxoplasma gondii per injection intraperitoneally, serum samples were collected and extracted DNA from serum samples at weeks0, 1, 2, 3, 4, 5, 6 and 7 for amplification of Toxoplasma gondii of specific gen by PCR.6, Injected with prednisolone into hind medial subcutaneous and with Azithromycin intraperitoneally after 5 Weeks infection, we observed PCR results and mouse mortality results and the number of brain cysts results.Results:1, The purify of our purified cysts is relatively high, inoculating the cysts can cause corresponding symptoms of toxoplasmosis in the susceptible mice in short time.2, ICR strain mice infected with 10 cysts all can spent about 2 weeks of the acute infection phase and survive.3, Comparison of sensitiveness of B1 gene primer and AF146527gene primer, we contrive that sensitiveness of B1 gene primer and AF146527gene primer are 0.16 pg, 16 pg, respectively.4, Through compared Lysis buffer method and DNA extraction kit method to extract DNA from whole blood of mice Toxoplasma gondii infection in the acute stage, we find that Lysis buffer method can extract Toxoplasma gondii DNA, but DNA extraction kit method can't.5, The method of PCR could amplify the products of Toxoplasma gondii of specific B1 gen before 5 weeks after challenge, while it couldn't 5 weeks after challenge.6, The infected mice all were death at between weeks 2 after injection Prednisolone (week 7),while the two infected mice of injection Prednisolone plus Azithromycin were only death at the same time (P <0.05), respectively. The number of cysts in brain tissues of mice injection Prednisolone was significantly increased (P<0.01).Conclusion:1, After improving the previous methods, we have successfully purified Toxoplasma gondii cysts. 2, we have established the model of infection of Toxoplasma gondii in ICR strain mice. Generally, the mice were infected intra-abdominally with 10 cysts.3, The B1 as target gen was more suitable for diagnosis of Toxoplasma gondii by PCR.4, Lysis buffer method is more suitable than DNA extraction kit method for extraction DNA from whole blood of mice toxoplasma infection in the acute stage.5, PCR method can accurately diagnosed toxoplasmosis in 5 week infection, but the mice were from acute stage to latent stage and PCR method can't diagnose toxoplasmosis after 5 week infection.6, Prednisolone could induce relapse of Asymptomatic Toxoplasma gondii Infection of mice and finally death caused by;Azithromycin is effective but it can not completely cure the Toxoplasma gondii infection.